Project Summary Hearing in mammals is dependent upon the ability to efficiently conduct sound vibrations from the environment to the inner ear. This conduction apparatus includes the auricle, external auditory canal, tympanic membrane (TM), middle ear space, and ossicular chain. Although a great deal of research has been directed to the biophysical properties of the ear less is known about the cellular composition of these structures and how the diverse cells that make up these structures are formed, maintained, and interact in pathological states. The TM has three layers: an outer layer of stratified squamous epithelium, a middle layer of connective tissue, and an inner layer of mucosal epithelium. It is unknown how many different cell types are present in each of these layers, where the stem or progenitor cell populations of these layers reside, or the dynamics of how these layers are maintained. Classic dye studies indicated that the outer epithelium of the TM migrates radially outward from the malleal attachment to the TM. We and others have shown that within the TM the vast majority of the proliferation is occurring near the malleus, and that cells then migrate radially outward. This implies at least two populations: a stem/progenitor population near the malleus, and a progeny population within the radial portions of the TM. We will first dissociate normal and injured TMs from mice and humans, sort cells, and perform single cell RNA sequencing analysis combined with nearest-neighbor clustering analysis using a novel algorithm, CellfindR, in order to identify the cellular subpopulations within the TM and to predict lineage relationships between them. We will confirm these populations by using immunofluorescent staining of mouse and human TMs. We will then perform pulse-chase labelling experiments using EdU as well as lineage tracing with genetically modified mice to validate the lineage hierarchies predicted by the psuedotime analysis, and definitively identify the stem and progenitor populations of the TM during perforation repair. Finally, we will perturb the PDGFR and BMP signalling pathway in defined populations of the TM using inducible and cell specific knockout mice in vivo as well as defined small molecule inhibitors in a novel ex vivo air-liquid interface model of explanted mouse and human TMs in order to define the mechanisms by which these populations differentiate and are maintained and to provide a therapeutic proof of concept. We hope that once we gain a deeper understanding of the functional cellular architecture and physiology within the TM, we can then learn how these processes go awry in and create better biological and surgical treatments for disorders of the TM.